Gary Felsenfeld: untangling chromatin’s mysteries

From DNA structure to gene expression, Felsenfeld has done a lot during his five decades at the NIH.

This year marks Gary Felsenfeld's 50th at the NIH, an institution he credits with much of his success.

It would be understandable if someone as accomplished as Gary Felsenfeld decided to take it easy and enjoy all his past successes, but this distinguished 81-year-old investigator is not one to rest on his laurels.

Sitting at his desk, which like most surfaces in his office is covered with stacks of papers, Felsenfeld recounts his group's most recent results with the enthusiasm of a graduate student who has just published his first article and not a scientific elder statesman who has more than five decades of influential discoveries under his belt.

It's easy to understand his eagerness to discuss the new findings though. Felsenfeld, currently chief of the section on physical chemistry in the Laboratory of Molecular Biology at the National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health, describes an intriguing mechanism by which the insulin gene can stimulate the expression of a far distant gene, sequentially speaking. This is accomplished by having insulin and the target gene brought into close physical proximity by external factors.

This long-distance regulation offers just one example of how researchers like Felsenfeld are changing the way we view the relationship between chromatin structure and gene expression. This sort of right-place–at-the-right-time understanding of gene regulation also may be an apt analogy for appreciating Felsenfeld's own scientific story.

Which kind of doctor?

While Felsenfeld's mentors during his training read like a "Who's Who" of 20th-century biochemists, his progression along the research path was not immediately obvious. His fascination with the natural world was cemented by the time he was eight, but his family interpreted his early interests as a sign that he would be a physician.

"At that time, if you were interested in biology, you had to be a doctor," he recalls. "I even remember my father, an attorney, telling me that science was just a hobby."

"He was right," Felsenfeld adds with a laugh. "Science is a fun hobby for me – one I'm paid to do!"

As a teenager, Felsenfeld was accepted to the renowned Stuyvesant High School, one of three specialized science high schools in New York City, and was a Westinghouse Science Talent Search finalist (now the Intel Science Talent Search). The finalists traveled to Washington, D.C., and toured the NIH, which Felsenfeld remembers being rather bleak and imposing. But he met young scientists who encouraged him to pursue his scientific interests.

Felsenfeld went on to attend Harvard University with the intent of becoming a physician and studied with John Edsall, the famous protein chemist and former Journal of Biological Chemistry editor. Edsall, who had gone to medical school but found it disappointing and instead became one of the earliest biophysical chemists, encouraged his scientifically inclined students to forgo medical training and get a doctorate instead.

Edsall's encouragement and a lackluster freshman biology course pushed Felsenfeld toward a more chemistry-oriented curriculum. In his last three years at Harvard, during weekly meetings, he and Edsall read and discussed influential scientific literature of the time including Pauling's "The Nature of the Chemical Bond" and Eyring, Walter and Kimball's "Quantum Chemistry."

"It made an enormous difference in my life," he says of those meetings, and the influence on his future career is evident.

During Felsenfeld's senior year, Linus Pauling gave a speech in which he said that anyone who chose chemistry as a career should take vows of poverty, an acknowledgement of the limited amount of funding and jobs for research scientists in those days. Interestingly, that only emboldened Felsenfeld. "I thought that forgoing monetary gain was wonderful, it was noble. And I committed myself to it."

Perhaps it was a bit of youthful naïveté on the part of a 20-year old student, but Felsenfeld, with Edsall's support, followed through and applied to graduate school at the California Institute of Technology to study physical chemistry. Edsall had told him that the future of biology was in chemistry, so Felsenfeld went to Caltech to learn theoretical chemistry and prepare himself for the new biology to come.

Out of respect for his parents, he also applied to Harvard Medical School, but did so having already made up his mind to get a doctorate degree. In an act that reveals his mischievous side, he sent Harvard a rejection letter to let them know of his decision.

During his second year at Caltech, Felsenfeld started working with Pauling, who had an interesting approach to helping students develop projects. "He'd leave notes in your mailbox with different ideas, and you'd find one that appealed to you." What appealed to Felsenfeld, and what would become the topic of his thesis, was the theory of ferromagnetism, although he readily admits, "I'm not sure I would understand it at this point!"

He completed his graduate work in three years and told Pauling he would like to move into biology. He wanted a position in Copenhagen with Kaj Linderstrøm-Lang, a prominent protein chemist, but Pauling said he needed more chemistry training and refused to write a recommendation. Instead, he wrote a recommendation for Felsenfeld to go to Oxford University to work with noted mathematician and theoretical chemist C.A. Coulson.

Although it sounds odd today, young scientists expected this level of guidance back then. "The idea that graduate students were people with rights had not yet really emerged," Felsenfeld says with a quiet laugh. All joking aside, he has no doubt about the wisdom of Pauling's decision.

"I was grateful. Pauling said 'I think this is what's right for you,' and I appreciated his guidance."

Returning stateside

Felsenfeld had a productive year with Coulson, predicting one of the earliest molecular structures using crystal field theory (the chlorocuprate anion CuCl42-) and completing what would be his last purely theoretical work.

He returned to the United States in 1956 and took up a post at the NIH, following an arrangement he had made prior to departing for Oxford. His draft board (the Korean War was over, but the draft was still active) had told Felsenfeld to obtain an officer's commission while in England or he would be serving in the infantry when he returned. Fortunately, Alexander Rich, whom Felsenfeld had befriended at Caltech, invited him to join the NIH as an officer in the Public Health Service.

Together with Rich and David Davies, another former Caltech colleague, Felsenfeld began working with synthetic polynucleotides, in vitro synthesized RNA segments of defined sequence, that in a few years would prove instrumental in cracking the genetic code.

Felsenfeld, Davies and Rich, though, were using these building blocks to understand how nucleic acids formed stable ordered structures like the recently solved DNA double helix. This was his first project with nucleic acids – and it proved to be an auspicious start.

While analyzing the salt requirements for double helix formation using complementary strands of poly-adenine and poly-uracil, Felsenfeld noticed that his spectrophotometer readings displayed some unusual absorption data at certain salt concentrations. Initially he tried to ignore it – perhaps he had made some experimental errors – but eventually he accepted that the data, which suggested a helix with twice as many U's as A's, was real. He remembers asking Davies, "Is there any way to fit a second poly-U into the structure?"

At the NIH, Felsenfeld, Davies and Rich uncovered the formation of a triple nucleotide helix.

What they had uncovered was the formation of a triple nucleotide helix. "It was a wonderful, wonderful moment, exhilarating. You're so lucky to have something like that when you're just starting out."

To be young and in science

After three years at the NIH, Felsenfeld was offered a faculty position in the biophysics department at the University of Pittsburgh. Biophysics was still emerging as a distinct field, and it was an unusual opportunity to join a discrete biophysics department. Recently married and ready to set out on a new endeavor, he accepted their offer. While continuing his biochemical characterizations of synthetic polynucleotides, Felsenfeld also was given leeway to start working on the copper protein hemocyanin, which he felt would be an ideal project to weave together his scientific interests in quantum chemistry and biology.

However, only two years later, the NIH brought Felsenfeld back to Bethesda with an offer he couldn't refuse. The intramural research director of the Institute of Arthritis and Metabolic Diseases (now NIDDK), DeWitt Stetten, had been persuaded to form a new laboratory of molecular biology. In a twist from the norm, this new group would be composed entirely of young, rising investigators rather than established scientists.

Felsenfeld recalls those early years as a marvelous time, and the lab was full of energy and enthusiasm. The only downside was that Felsenfeld quickly realized that his studies with nucleic acids would be all-consuming; after a few years, the work with hemocyanin fell by the wayside.

He continued investigating the stabilization of multistranded nucleic acid structures by counter ions; this soon led to studies using polylysine and polyarginine as models to examine the interaction of DNA with basic proteins in the nucleus. Eventually, though, he got tired of saying they were good models, "because they weren't good models!"

So he decided to work directly with chromatin. The move resulted in a big change for the longtime chemist. "The thing with chromatin," he says, "is that I got sucked into the biology and trying to figure out what is the biological function of this DNA-protein packaging."

Insulated activity

Felsenfeld's early work in chromatin biology was aimed at understanding how a gene is controlled through a combination of histone interactions and transcription factors. He used the four-gene chicken beta-globin gene locus for his studies, which was an ideal system for his work, because chicken blood cells have stable chromatin that can be isolated easily in large quantities. Using the beta-globin model, his group contributed numerous findings regarding the role of structural and biochemical changes in chromatin in regulating globin gene expression.

Later, Felsenfeld became more intrigued at what lay at the edges of the beta-globin locus. In blood cells, the globin locus is an open and accessible chromatin domain; at the terminus, where the locus borders a stretch of condensed chromatin, there is a DNase hypersensitive site that appears to mark the boundary. (HS sites are short regions of chromatin distinguished by their extreme sensitivity to nuclease cleavage.)

Felsenfeld proposed testing this region to determine if it did, in fact, constitute a boundary between the open and closed domains. At the time, there was only one published example of such an insulator element, the gypsy element in Drosophila. Victor Corces and Dale Dorsett had shown that gypsy could block an enhancer's ability to increase gene promoter activity if positioned between the two, effectively insulating the promoter from enhancer influence. Felsenfeld's group tested the HS site and found that it behaved similarly.

They commenced studying the insulator region in detail and discovered several protein binding sites, one of which, they showed, bound a protein that was necessary and sufficient for enhancer blocking. The protein, CTCF, had been known for some time to regulate gene activity; however, this new discovery suggested it might also be involved in higher order chromatin organization.

They looked for other locations where CTCF might function and found that it played a critical role in the control of the Igf2/H19 imprinted locus. This two-gene region is special in that individuals only express the paternal copy of Igf2.

Felsenfeld's group described a regulatory mechanism in which the H19 and Igf2 genes are separated by an imprinted control region containing CTCF-binding sites. The ICR on the paternal allele is methylated, preventing CTCF from binding and allowing a downstream enhancer to promote expression of both genes. The maternal allele, however, remains unmethylated and capable of binding CTCF, thus blocking enhancer activity and preventing it from driving expression of Igf2. Similar results were obtained independently in the laboratories of Shirley M. Tilghman at Princeton University and Rolf Ohlsson at the Karolinska Institutet in Sweden.

The role of CTCF now is well established, and it's been shown to function by promoting the formation of DNA loops that bring distant genetic elements physically closer. "CTCF is part of a regulatory network that's three dimensional and physical, long-range physical," Felsenfeld says enthusiastically. "We just keep going up in scale."

A possible mechanism by which an insulator element could protect against the propagation of a silencing histone modification into a transcriptionally active chromatin domain.

Here and Now

Felsenfeld is continuing his own upward trajectory, and recently his group began working on human pancreatic cells. Given that the insulin gene is close to the imprinted Igf2/H19 locus, Felsenfeld has become interested in potential long-range contacts between insulin and other genes mediated by CTCF, and that has led to his most recent findings that the insulin gene's physical proximity with a distant gene's regulatory elements affects that target gene's expression.

Imprinting at the IGF2/H19 locus: presence of a CTCF-dependent insulator.

Despite the rapidly changing nature of his work, Felsenfeld has managed to keep a proper focus on the "big picture," an ability that arises from a keen intellectual discipline honed over many years and mentors. "Always keep in mind what you are trying to answer," he says. "Something may seem interesting, but if it's not directly relevant, note it and hope to remember that it exists, but you have to move on.

"Edsall once said, 'I stop outside the atomic nucleus; I've got enough to think about,'" Felsenfeld recalls. "And it's true. You can only do so much!"

In Felsenfeld's case, though, only so much seems to be a lot. This year marks the 50th year of his lab at the NIH, an institution he credits with much of his success. "The NIH Intramural program is one of the few places in the world where I could do science the way I wanted to."

Along the way, Felsenfeld has had the fortune to have great people around him. Foremost would be his family (including three children and eight grandchildren), which has long been a pillar of support. And of course, all the work carried out over those 50 years would not have been possible without a remarkable group of postdocs and grad students. Many of his former protégés now are distinguished researchers in their own right, which provides great pride for Felsenfeld, who considers training young scientists to be one of his most important responsibilities.

And he shows no signs of slowing down.

"Whenever someone asks, 'What's the most exciting thing you've done?' I say, 'What we're doing right now,' because that's all that counts."